Human Genetics Lecture #1 March 25, 2014

Bio 5491 - Advanced Genetics

Cristina de Guzman Strong, Ph.D. Department of Medicine Dermatology/Pharmacogenomics McDonnell Basic Sciences 770 [email protected] 362-7695 Human Genetics

• Karyotype • Mutations in Regulatory • Genetic Variants Elements • Mendelian Diseases • Copy number variation • Linkage, positional cloning diseases • Penetrance/Expressivity • Mitochondrial genetics • Human Genome Project • Human-specific variation • GWAS/Next-Gen • Future sequencing (Exome) • Undiagnosed Diseases • Epigenetics/ENCODE What is Human Genetics?

The relationship between natural DNA sequence variation(s) and human phenotypic traits What is different about Human Genetics?

• Imprinting……..uniquely mammalian.

• Trinucleotide repeat diseases…….anticipation.

• One can study complex behaviours and cognition.

• Extensive sequence variation leads to common/ complex disease 1. Common disease – common variant hypothesis 2. Large # of small-effect variants 3. Large # of large-effect rare variants 4. Combo of genotypic, environmental, epigenetic interactions Greg Gibson, Nature Review Gen 2012 Human Genome (Karyotype)

22 autosomes/ XY sex chromosomes

02_13.jpg

Human genome is ~41% GC, but that is non-randomly distributed. Dark G-bands are lower GC (and lower gene content)

Genetic variation: Single Base Pair C T

– SNP (single nucleotide polymorphism) - Freq > 0.01 – Can also be 1 insertion or 1 deletion, “indel” – Alleles with Freq < 0.01 – called rare variants OR SNPs – Mutations: usually, really RARE. Alter protein function or regulation. Simple Mendelian Trait. Can cause disease

Understanding the SNP Major vs. Minor Allele

• Referring to coding OR non-coding region • Nomenclature – rs”X” (SNP), can also be indel Looking up a SNP

C T Looking up a SNP

C T Types of Coding Mutations

K867X K867R K867T Courtesy of Laura Elnitski Example of Nomenclature (NF1)

Ars et al., HMG 2000 Resource for Sequence Variant Calling • http://www.hgvs.org/mutnomen/recs.html Genetic Variation: Copy Number Insertion/deletions of small number of bp (indels): – Polymorphism - also known as copy number variation (CNV) – Indels in coding region – frameshift mutation if less than 3 bp – Repeat sequences: di (TG), tri, or tetra – microsatellites

Larger repeats: VNTRs (variable number of tandem repeats), minisatellites - used for forensics

Deletions or duplications of larger blocks of DNA encompassing one or more genes (Williams or DiGeorge Syndrome)

Genetic Variation: Copy Number Larger repeats: VNTRs (variable number of tandem repeats), minisatellites - used for forensics

Description of diseases and mutations • Online Mendelian Inheritance in Man (McKusick): http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=OMIM

Human mutation database : http://www.hgmd.org Characteristics of Simple/

Mendelian diseases • >20,000 Mendelian traits described in OMIM (Online Mendelian Inheritance in Man) • Freq usually < 1/10,000 • Most mutations: Loss of function and recessive phenotypes, e.g. inborn errors of metabolism • Dominant mutations: Cause disease due to 50% of protein product (haploinsufficiency) or dominant negative effect • Some dominant mutations lead to “gain of function” - e.g. expanded polyglutamine repeat leading to abnormal aggregate (triplet repeat diseases) Thompson & Thompson, Genetics in Medicine (5th) Basic Mendelian pedigree patterns 04_02.jpg Example of Mendelian disorder: Waardenburg Syndrome (PAX3: Deafness in association with pigmentary anomalies and defects of neural crest-derived tissues). Loss of function mutations in PAX3 gene: Type 1 Waardenburg syndrome 16_01.jpg Variable penetrance versus variable expressivity Penetrance - the frequency of expression of an allele when it is present in the genotype of the organism Example: if 9/10 of individuals carrying an allele express the trait, the trait is said to be 90% penetrant

Expressivity - variation in allelic expression when the allele is penetrant. Example: For polydactyly, an extra digit may occur on one or more appendages, and the digit can be full size or just a stub. Modifier genes can affect penetrance, dominance, expressivity Neurofibromatosis ( NF1)

• Mutations in neurofibromin gene • Reduced penetrance • Wide range of symptoms – variable expressivity

Nature 455, 1061-1068(23 October 2008) Cloning of Genes for Simple Mendelian Traits where the biochemical defect is not known

Linkage (to locate chromosomal region), followed by positional cloning Standard of Proof for Positional Cloning

For fully penetrant, simple Mendelian traits one usually detects obviously deleterious mutations in coding regions for multiple alleles. Positional Cloning 14_02.jpg Characteristics of families for Linkage Analysis

Large number of affected members required (generally > 10):

Single large families where LOD of theta > 3 OR Multiple small families: Add LOD scores at different theta values to obtain LOD > 3

Example of positional cloning: Huntington’s disease

Dominantly inherited Fatal presenile dementia. Symptoms include spasmodic jerking of the limbs, psychotic behavior, and mental deterioration.

First described by George Huntington in 1872. Incidence is 5-10/100,000.

Genetic linkage to chr 4: 1984: Nancy Wexler, Jim Gusella

1983 - Linkage to G8 (also called D4S10) Mapped approximately 4 cM from the HD locus. 10 more years to clone the HD to clone gene.

Huntingtin gene - HTT Haplotype analysis 14_04.jpg How did we get here? • ~1,200 “simple” Mendelian trait loci have been cloned, mostly by genetic linkage analysis and positional cloning. • The revolution in human genetics has been driven by advances in genomics, DNA sequencing and polymorphism detection. Courtesy of Eric Green Courtesy of Eric Green Post HGP 1000 Genomes Project

sequence about 2,000 unidentiﬁed individuals from 20 populations around the world Capture minor allele frequencies as low as 1% Post HGP Published Genome-Wide Associations through 12/2012 Published GWA at p≤5X10-8 for 17 trait categories

NHGRI GWA Catalog www.genome.gov/GWAStudies www.ebi.ac.uk/fgpt/gwas/ 1% of the genome – 30Mb From Nimblegen Slide courtesy of Elliott Margulies

Exome sequencing – XIAP Whole Bone Marrow Transplant January 2010 Slide courtesy of Eric Green Slide courtesy of Eric Green (modiﬁed) Slide courtesy of Eric Green Slide courtesy of Eric Green Hon, Hawkins, Ren review (Human Molecular Genetics 2009)

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Maher, Nature 2012 Mutations in regulatory regions

– Altered TF binding to promoter or enhancer e.g. sonic hedgehog enhancer – Splice site alterations (e.g. some beta thalassemias) – Altered mRNA stability (e.g. in AAUAA) – Altered micro RNA binding leading to altered protein translation Sequence variants in SLITRK1 are associated with Tourette's syndrome. Science 2005, 310.

Nature March 23 2011 Advance issue

Copy Number (Structural) Variation Structural variation of the genome kilobase- to megabase-sized deletions, duplications, insertions, Inversions complex combinations of rearrangements. CNVs due do non-allelic recombination between low-copy repeats (LCRs) that lead to human disease 11_19_2.jpg

(Smith-Magenis syndrome)

DiGeorge Syndrome

Velocardiofacial syndrome Examples of contiguous gene syndromes • Xp: – Successively large deletions remove more genes and add more diseases • 11p12: – WAGR (Wilm’s tumor, Aniridia, Genital and/or urinary tract abnormalities, Mental retardation) Segmental aneuploidy

• Phenotype in heterozygotes depends on only a subset of deleted genes that are dosage sensitive • De Novo microdeletions, frequently flanked by long repeats (often transcribed hence open chromatin - more recombination prone)

1/20,000 births Growth retardation Hypercalcemia Sypervalvular aortic stenosis (elastin) Moderately mentally retarded Highly sociable, often musical, defect in visuospatial constructive ability Deletions identified in Williams syndrome Current knowledge of of CNV (copy number variation) in the human genome http://projects.tcag.ca/variation/ 011/10: Total entries: 101,923 (hg18) CNVs: 66,741 Inversions: 953 InDels (100bp-1Kb): 34,229 Total CNV loci: 15,963 Articles cited: 42

CNV afecting one human phenotype: Ability to digest starch:

Diet and the evolution of human amylase gene copy number variation

Perry et al. Nature Genet. Nature Genetics 39, 1256 - 1260 (2007) Different types of mechanisms leading to SVs/CNVs

• Non-allelic homologous recombination (NAHR) • Non homologous end joining (NHEJ) • Transposon insertion (e.g. L1) Gu et al. PathoGenetics 2008 1:4 Genomic rearrangements in the genome are likely to be more common than expected 5% of human genes are found in interspersed duplicated copies Recent examples of large rearrangement polymorphisms in the human genome • Sebat et al. Large-scale copy number polymorphism in the human genome. Science 305:525-8 (2004) • A common inversion under selection in Europeans Stefansson et al. Nature Genetics 37, 129 - 137 (2005)

Chromosome 17q21 H1

H2 900kb A common inversion under selection in Europeans Stefansson et al. Nature Genetics 37, 129 - 137 (2005)

CRH1 MAPT

20% chromosomes

900kb

CRH1: corticotropin releasing hormone receptor 1 MAPT: microtubule associated protein tau Genomic architecture, rearrangements and marker genotypes at 17q21.31 Microdeletion syndromes: 1.Koolen, D.A. et al. Nat. Genet. 38, 999– 1001 (2006). 2.Shaw-Smith, C. et al. Nat. Genet. 38, 1032–1037 (2006). 3.Sharp, A.J. et al. Nat. Genet. 38, 1038– 1042 (2006). Affected individuals with 17q21 microdeletion (1% of mental retardation patients) Fragile X – trinucleotide repeat

• Most common inherited form of mental retardation • Due to instable CGG repeat at FMR1 • All full mutations derive from premutation (56-200 repeats) • Expansion through female meiosis • Severity correlates with CGG repeats Rare L1 insertions as a cause of disease LINEs (long interspersed repetitive elements)

AKA: Kpn1 element ~5kb. Relic of retrovirus 3 distantly related LINE families are found in the human genome, but only LINE1 is active. Human genome: ~515,000 copies of LINE1 (L1), ~365,000 L2, and ~37,000 L3 (most are truncated or rearranged) Only ~30-60 are active In mouse, ~3,000 are active. L1 Insertions Occasionally lead to genetic disorder: Reported in Duchenne muscular dystrophy, type 2 retinitis pigmentosa, thalassaemia, chronic granulomatous disease, and hemophilia A (2/140 patients) - insertion into factor VIII.

Prak & Kazazian. (2000) NRG. 1: 134-144 L1 elements also cause Transduction.

In addition to duplicating themselves, L1s can carry with them genomic flanking sequences that are downstream of their 3UTRs.

Prak & Kazazian. (2000) NRG. 1: 134-144 Mitochondrial genetics Mitochondrial inheritance gives matrilineal pedigree pattern

• Matrilineal inheritance • Sperm mtDNA is actively degraded • Mitochondria present in thousands of copy per somatic cell • Normal individuals: ~99.9% of molecules are identical (homoplasmy) • New mutation leads to heteroplasmy • In some patients with mitochondrial disease every patient carries causative mutation (homoplasmy) • In some patients there is heteroplasmy. The mitochondrial genetic bottleneck Mitochondrial genome

• Cytoplasmic organelle • Small genome (~16kb) • 1/200,000 size of nuclear genome • Sequenced in 1981 (Anderson et al.) • 93% is coding • ~ 1,000 copies per cell Organ systems and symptoms of mt diseases

• Multisystem disorders with large range of symptoms: • Brain, heart, skeletal muscle, kidney and endocrine systems can be affected (sometimes there is a threshold effect) • Symptoms: forms of blindness, deafness, movement disorders, dementias, heart disease, muscle weakness, kidney dysfunction, endocrine disorders (including diabetes) Functions of the 37 mitochondrial genes • 13 of mitochondrial peptide subunits in mitochondrial respiratory-chain complex (OXPHOS) – Remaining > 67 OXPHOS subunits are nuclear encoded • rRNAs: 2 • tRNAs: 22; Located between every 2 rRNA or Protein coding genes • Third base wobble • All factors involved in maintenance,replication & expression of mtDNA are Nuclear encoded Example of moderately severe tRNA mutations tRNA Lys : A8344G (Frequent) T8356C G8363A G8361A MERRF (Myoclonic Epilepsy and Ragged-Red Fiber Disease) Onset: Late adolescence - Early adult Level of mutant heteroplasmy + age of patient influence severity of symptoms

Mitochondrial DNA and human evolution: Nature 325, 31 – 36 (1987) Rebecca L. Cann*, Mark Stoneking & Allan C. Wilson

Mitochondrial DNAs from 147 people, drawn from five geographic populations have been analyzed by restriction mapping. All these mitochondrial DNAs stem from one woman who is postulated to have lived about 200,000 years ago, probably in Africa. All the populations examined except the African population have multiple origins, implying that each area was colonized repeatedly J lineage often seen with Leber’s hereditary optic neuropathy (LHON) patients Identifying Human Variation

hCONDEL – loss of enhancer for 1) sensory vibrissae, 2) penile spine enhancer in androgen receptor, loss of enhancer in tumor suppressor gene HAR1 – part of noncoding RNA expressed in human brain that has undergone expansion

HAR2 – enhancer involved in opposing thumb February 2011